1

Supplementary Information

A 3.4-Å cryo-EM structure of the human coronavirus spike trimer computationally derived

from vitrified NL63 virus particles

Kaiming Zhang1, Shanshan Li1, Grigore Pintilie1, David Chmielewski2, Michael F. Schmid4,

Graham Simmons3, Jing Jin3 and Wah Chiu1,4,*

1Department of Bioengineering and James H. Clark Center, Stanford University, Stanford, CA

94305, USA 2Graduate Program in Biophysics, Stanford University, Stanford, CA 94305, USA 3Vitalant Research Institute, San Francisco, CA 94030, USA 4Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Menlo

Park, CA 94025, USA

*Correspondence: wahc@stanford.edu

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Fig. S1. Single-particle cryo-EM data processing of HCoV-NL63 coronavirus spike glycoprotein.

(a). Workflow of the data processing. (b) Gold standard FSC plot for the final 3D reconstruction.

(c) Euler angle distribution of the particle images of computationally extracted spikes, calculated

in cryoSPARC. (d) Cross-correlation coefficient between the 3D reconstructions with (green) and

without (grey) C3 symmetry applied.

MotionCor2 & Patch CTF Estimation

Manually Curate Exposures

4,138 movie stacks

4,129 micrographs

Manually picking (157 particles)

2D classification

Template auto-pick

944,822 particles

Ab-initio 3D reconstruction

2D classification

300,236 particlesSelection of

representative classes41,548 particles

15.2% 62.2% 22.6%

Heterogeneous Refinement

(3 rounds)

Non-Uniform Refinement

82,030 particles

With C3 symmetryWithout symmetry

Two reference maps

CTF Refinement

3.45 Å

Non-Uniform Refinement

3.66 Å 3.39 Å

a

b

c

dCC = 0.98

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Fig. S2. Structure-based alignment of domains of spike protein from HCoV-NL63 and SARS-

CoV-2. The method of TM-align was used, yielding good structural alignments illustrated in Fig.

5. Aligned sequences along with RMSD, percent identity, and TM-score are shown for all domain

pairs.

CoV2 Domain A’ to NL63 Domain 0 CoV2 Domain A’ to NL63 Domain A

NL63 Domain A to NL63 Domain 0 CoV2 Domain B’ to NL63 Domain B

CoV2 Domain C’ to NL63 Domain C

CoV2 Domain D’ to NL63 Domain D

CoV2 Domain S2’ to NL63 Domain S2

Aligned length=159, RMSD=3.48Å, Identity (#identical/#aligned)=7.5%TM-score= 0.58993 (if normalized by length of Chain_1, i.e., LN=203, d0=5.30)TM-score= 0.59225 (if normalized by length of Chain_2, i.e., LN=202, d0=5.29)

(":" denotes residue pairs of d < 4.0 Angstrom, "." denotes other aligned residues)

CoV2_A AYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLD:.........:::::::::::::: . ...::. ::::::: :::::...::::: ::::::..:

NL63_0 -------ANLSMLQLGVPDNSSTIVTGLLPT--H-WFCANQ--STSVYSA--NGFFYIDVGNHRS-AFALHTGYY

CoV2_A SK-TQSLLIVNNAT---NVVIKVCEFQFCNDPFLGV---NCTFEYVSFK---NLREFVFKNIDGYFKIYSKHTPI.: ::::::::... .:::::::::.. :::::::. ...::::::::::::::.

NL63_0 DANQYYIYVTNEIGLNASVTLKICKFSRS-------SSFDCIVNLLF--TEQLGAPLGITISGETVRLHL-----

CoV2_A NLVRDLPQGFSALEPLVDLPIG-I-NITRFQTLLALH--A-AYYVGYLQPRTFLLKYNENGTITDAVD-------....:::::: : :::::::... : ....::::::::::::::: :::::::

NL63_0 ------------YNVTRTFYVPAAYKLTKLSVKCY--FNYSCVFSVVNATVTVNVTTHNG-RVVNYTVCDDCNGY

CoV2_A ---------------------

NL63_0 TDNIFSVQQDGRIPNGFPFNN

Aligned length=157, RMSD=3.72Å, Identity (#identical/#aligned)=10.2%TM-score= 0.57667 (if normalized by length of Chain_1, i.e., LN=203, d0=5.30)TM-score= 0.56989 (if normalized by length of Chain_2, i.e., LN=206, d0=5.34)

(":" denotes residue pairs of d < 4.0 Angstrom, "." denotes other aligned residues)

CoV2_A AYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIH-DNPVLPF-----------------NDGVYFAS.:.:.:.:.....:::::::::.:. .::.:::.. .:::::: .:::::::

NL63_A -------NWFLLTNGSTLVDGVSRLYQPLRLT-CLWPVPGLK-SSTGFVYFNATGSDVNCNGYQHNSVVDVMRYNL

CoV2_A TEK----SN-IIRGWIFGTTLDSKTQSLLIVN---------N-A-----T-NVVIKVCEFQFCNDPFLGV-NCTFE::. :: :..::::::. .:::::::. : . . ::::::::.. ....:

NL63_A NFSANSLDNLKSGVIVFKTL---QYDVLFYCSNSSSGVLDTTIPFGPSSQPYYCFINSTIN---------TTHVST

CoV2_A YV-SFK---NLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALH--AAY-YVG:. . ..::::::.. . ::::: :: :::::: ::::::::::.. ::: :::

NL63_A FVGI--LPPTVREIVVART-G-QFYIN------------------GF-KYFDLG-FIEAVNFNVTTASATDFWTVA

CoV2_A YLQPRTFLLKYNENGTITDAVD--:::::::::::. ....:::::

NL63_A FATFVDVLVNVS-ATNIQNLLYCD

Aligned length=160, RMSD=3.88Å, Identity(#identical/#aligned)=8.1%TM-score= 0.54596 (if normalized by length of Chain_1, i.e., LN=206, d0=5.34)TM-score= 0.55398 (if normalized by length of Chain_2, i.e., LN=202, d0=5.29)

(":" denotes residue pairs of d < 4.0 Angstrom, "." denotes other aligned residues)

CoV2_B NWFLLTNGSTLVDGVSRLYQ-PLRLTCLWPVPGLKSSTGFVYFNATGSDVNCNGYQHNSVVDVMRYNLNFSANSLD.::.... ....:::.:::: .... :..:::.: :::::: ..::: :.:

NL63_B ANLSMLQ-LGVPDNSSTIVTGLLPT--HWFCANQS--TSVYSA------------------NGFFY-IDV------

CoV2_B NLKSGVIVFKT---L--QYDVLFYCSNSSSGVLDTTIPFGPSSQPYYCFINSTIN---TTHVSTFVGILPPTVREI::::::::::. . ::::::: .:: .:..:.:::.::.. .:.:.:::.:::::::::

NL63_B GNHRSAFALHTGYYDANQYYIYVT-----------NEI---GLNASVTLKICKFSRSSSFDCIVNLLFTEQLGAPL

CoV2_B VVART---GQFYINGFKYFD-L--GF-IEAVNFNVTTASATDFWTVAFATFVDVLVNVSATNIQNLL-Y-CD----:::.: ::::::.::..: . :: :::::::. . .:.::..:.:..:....::::.:::::.. . .:

NL63_B GITISGETVRLHLYNVTRTFYVPAAYKLTKLSVKC-Y-FNYSCVFSVVNATVTVNVTTHNGRVVNYTVCDDCNGYT

CoV2_B --------------------

NL63_B DNIFSVQQDGRIPNGFPFNN

Aligned length=95, RMSD=4.13Å, Identity (#identical/#aligned)=9.5%TM-score= 0.38757 (if normalized by length of Chain_1, i.e., LN=168, d0=4.83)TM-score= 0.44403 (if normalized by length of Chain_2, i.e., LN=140, d0=4.40)

(":" denotes residue pairs of d < 4.0 Angstrom, "." denotes other aligned residues)

CoV2_B -ITNLCPFGEVFNAT---------------RFASVYA---WNRKRISNCVADYSVLYNS---------------AS:::::: : ::.. ::. ..::::::.:::. .. ..

NL63_B YYQHTDI-N--FTATASFGGSCYVCKPHQVNIS----LNGNTSVCVRTSHFSI-RY---IYNRVKSGSPGDSSWHI

CoV2_B FSTFKCYGVSPTKL-NDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKGNYN:.:::.:::....: ::::::::::::: ...: . ..:.:::..

NL63_B YLKSGTCPFSFSKLNNFQKFKTICFSTV----------------------EVPG--S-CNFPLEATW---------

CoV2_B YLYRKPFERDIYFPLQSYGFQPT---NVGYQPYRVVVLSFELLHAPATVCGPK-------K..: .:::::::::: : .: ::::. .

NL63_B -----------------------HYTSYT--IVGALYVTWSE-G-NS-ITGVPYPVSGIRE

Aligned length=59, RMSD=1.96Å, Identity (#identical/#aligned)=25.4%TM-score= 0.71304 (if normalized by length of Chain_1, i.e., LN=61, d0=2.64)TM-score= 0.72167 (if normalized by length of Chain_2, i.e., LN=60, d0=2.61)

(":" denotes residue pairs of d < 4.0 Angstrom, "." denotes other aligned residues)

CoV2_C STNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTL-EILDITPC::::::::::::::::::::::::::::::: :::::::.:.:::::::. .: ::::::::

NL63_C FSNLVLNNCTKYNIYDYVGTGIIRSSNQSLA-GGITYVSNSGNLLGFKNV-STGNIFIVTPC

Aligned length=63, RMSD=3.10Å, Identity (#identical/#aligned)=6.3%TM-score= 0.53671 (if normalized by length of Chain_1, i.e., LN=77, d0=3.11)TM-score= 0.58449 (if normalized by length of Chain_2, i.e., LN=68, d0=2.86)

(":" denotes residue pairs of d < 4.0 Angstrom, "." denotes other aligned residues)

CoV2_D SFGGVSVITPGTNTSNQVAVLYQDVNCTE-V----NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASY...::::::::: ::::::: :.:. . ::::::.:::::.:.:...: .::::: ::::::

NL63_D CNQPDQVAVYQQ---SIIGAMT--AVNE-SRYGLQNLLQLPNFYYVSNGGNNCTT-AVMTYS-NFGICA

CoV2_D QTSQSIIAYTMSL:::::::

NL63_D ----DGSLIPV--

Aligned length=416, RMSD=3.10Å, Identity (#identical/#aligned)=37.0%TM-score= 0.87726 (if normalized by length of Chain_1, i.e., LN=422, d0=7.39)TM-score= 0.80337 (if normalized by length of Chain_2, i.e., LN=464, d0=7.70)

(":" denotes residue pairs of d < 4.0 Angstrom, "." denotes other aligned residues)

CoV2_S2 --GAENSVA-YS-N-NSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTG::..... .. . :.::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NL63_S2 RPRNSSDNGISAIITANLSIPSNWTTSVQVEYLQITSTPIVVDCATYVCNGNPRCKNLLKQYTSACKTIEDALRL

CoV2_S2 IAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKP----SKRSFIEDLLFNKVT-------------:::::::.::..::.....:::...:.:.::::::::::.... .::::::::::::::

NL63_S2 SAHLETNDVSSMLTFDSNAFSLANVTSFGDYNLSSVLPQRNIRSSRIAGRSALEDLLFSKVVDVDYKSCTKGLSI

CoV2_S2 ------K-FNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQ: ::::::::::::::::::::::::::: ::. .. ....::::::::::::::::.:.:..::::

NL63_S2 ADLACAQYYNGIMVLPGVADAERMAMYTGSLIGGM-VLG-GL--TSAAAIPFSLALQARLNYVALQTDVLQENQ

CoV2_S2 KLIANQFNSAIGKIQDSLSS------TASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPE:::::::::::::::::::: ...:::::::::::::::::::::::::::::::::::::::::::::

NL63_S2 KILAASFNKAINNIVASFSSAEAIHTVTIALNKIQDVVNQQGSALNHLTSQLRHNFQAISNSIQAIYDRLDSIQ

CoV2_S2 AEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVF::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

NL63_S2 ADQQVDRLITGRLAALNAFVSQVLNKYTEVRGSRRLAQQKINECVKSQSNRYGFCGNGTHIFSIVNSAPDGLLF

CoV2_S2 LHVTYVPAQEKNFTTAPAICHD-GKAHFPRE--GVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIV:::::::::::::.:::::::: ::::::.. ::::......:::::::::::...:. :::::::: :.:::

NL63_S2 LHTVLLPTDYKNVKAWSGICVDGIYGYVLRQPNLVLYSDNGVFRVTSRVMFQPRLPVLS-DFVQIYNC-NVTFV

CoV2_S2 NN----T--VYDPLQPE--L--DS:. . ::::.... . ..

NL63_S2 NISRVELHTVIPDYVDVNKTLQEF

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Table S1. Cryo-EM data collection, refinement, and validation statistics. HCoV-NL63 S protein

(EMDB-22889) (PDB ID: 7KIP)

Data collection and processing Magnification 64k Voltage (kV) 300 Electron exposure (e–/Å2) 48 Defocus range (μm) -0.4 to -3.6 Pixel size (Å) 1.4 Symmetry imposed C3 Initial particle images (no.) 944,822 Final particle images (no.) 82,030 Map resolution (Å) FSC threshold

3.39 0.143

Map resolution range (Å) 2.7-7.0 Refinement Initial model used (PDB code) 5SZS Map sharpening B factor (Å2) -90 Model composition Non-hydrogen atoms Protein residues Ligands

30,612 3,576 222

B factors (Å2) Protein Ligand

94.84 141.21

R.M.S. deviations Bond lengths (Å) Bond angles (°)

0.009 1.30

Validation MolProbity score Clashscore Poor rotamers (%)

1.44 1.88 0

Ramachandran plot Favored (%) Allowed (%) Disallowed (%)

92.06 7.86 0.08

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Table S2. Comparison of the HCoV-NL63 S glycans in different studies.

Amino acid residue site Sequon

MS identified (Walls et al)

Cryo-EM observed (Walls et al)

Cryo-EM observed (this study)

24 NLSM ND ND 2435 NSST 35 35 3552 NQST 52 52 5298 NASV 98 98 98111 NTTF ND ND ND119 NASS ND ND ND155 NVTR 155 155 155178 NYSC ND ND ND187 NATV 187 187 187193 NVTT 193 193 193203 NYTV ND ND 203240 NGST 240 240 240276 NATG 276 276 276301 NFSA 301 301 301330 NSSS ND 330 330354 NSTI ND 354 354358 NTTH 358 358 358403 NVTT 403 403 403426 NVSA ND 426 426486 NFTA 486 486 486506 NISL 506 506 506512 NTSV 512 512 512626 NCTK 626 626 626645 NQSL 645 645 645666 NVST 666 666 666699 NESR 699 699 699723 NCTT ND 723 723749 NSSD ND 749 749762 NLSI ND 762 762768 NWTT 768 768 768844 NVTS 844 844 844852 NLSS 852 852 8521111 NGTH 1111 1111 11111196 NVTF 1196 1196 11961201 NISR 1201 1201 12011218 NKTL 1218 1218 12181242 NLTY 1242 ND ND1247 NLSS 1247 ND ND1277 NSTY 1277 ND ND

TOTAL # of sites39 28 31 33